The study of maturation stage-dependent expression of genes that are specific for the functions of neutrophils is relevant to better understand, mechanistically, myeloid blood cell differentiation, Since acute myelogenous leukemia can be considered to derive from a block in differentiation, such new information may provide more insights into the pathogenesis of this disease as well. Defensins (HNP) are microbicidal peptides and the principal constituents of human neutrophil granules. HNP-1 transcripts are selectively present in promyelocytic leukemia cells (HL-60) and unique temporal regulation occurs during drug-induced differentiation. A 165 bp minimal promoter was identified in the 5'- upstream region of the HNP-1 gene that directs HL-60-specific gene expression in-vivo; no consensus binding sites for known factors are present The main objective of this proposal is to study putative novel factors, or factor combinations, that control promyelocytic expression of defensin. To this end, a three-stage approach will be used. First, control elements will be precisely located, based on their ability to i) drive HL-60 specific expression of reporter genes in transient transfection assays, and ii) give specific band patterns in electrophoretic mobility assays (EMSAs). Second, the consensus recognition sites for the binding proteins will be determined by i) in- vitro footprinting using HL-60 nuclear extracts, and ii) site-directed mutagenesis and functional analysis (as in stage I). Finally, binding factors will be characterized by, in order, i) crosslinking and Southwestern blotting, ii) column chromatographic (EMSA assaying) and gelelectrophoretic purification, iii) microsequencing / mass spectrometric analysis of protein fragments, iv) identification and/or cloning of the cDNA, v) examination of cell specific expression, and vi) examination of transactivating potential in other cell types.